3-Acetoxymethyl 7-amino-3-cephem-4-carboxylic acid (7-aminocephalosporanic acid, 7-ACA) is the starting material for the synthesis of many semi-synthetic cephalosporin antibiotics. 7-ACA can be generated from cephalosporin C (a readily available fermentation product) by a two-step enzymatic process (Scheme 1), using first a D-amino acid oxidase in conjunction with oxidative decarboxylation to produce glutaryl 7-ACA (Step A), and then using a glutaryl 7-ACA acylase to remove the glutaryl group to produce 7-ACA (Step B). Steps A and B can also be carried out by chemical processes. The chemical processes, which involve the use of large quantities of organic solvents and toxic chemicals, have safety and environmental disadvantages. By using enzymatic processes, on the other hand, 7-ACA is obtained under mild conditions in an aqueous solvent system. Enzymes that catalyze the hydrolysis of glutaryl 7-ACA to 7-ACA (Step B) have been readily available, but enzymes that efficiently catalyze direct hydrolysis of cephalosporin C to 7-ACA (Step C) have not. Consequently, a two-step process has been generally employed, where step A is carried out chemically or enzymatically, and step B is carried out enzymatically. See for example Cambiaghi et al., U.S. Pat. No. 5,424,196, and references therein.
Step A is usually carried out with D-amino acid transaminases (also known as D-amino acid oxidase, EC-1.4.3.3) (Aretz et al., U.S. Pat. No. 4,745,061), in order to oxidize the amino group of the D-amino acid side chain to a keto group, followed by treatment with hydrogen peroxide to effect decarboxylation and provide glutaryl 7-ACA.
Much effort has gone into the development of efficient methods for carrying out Step B. Crawford et al., in U.S. Pat. No. 5,104,800, provide a brief summary of earlier work in the field of 7-ACA synthesis. Matsuda et al, in U.S. Pat. No. 3,960,662, describe glutaryl 7-ACA amidase activity from cultures of Comamonas sp. and Pseudomonas ovalis. Workers at Fujisawa Pharmaceutical Co. (Aramori et al., U.S. Pat. No. 5,310,659 and EP 0482844; Aramori et al., 1991, J. Bacteriol. 173:7848-7855) describe a glutaryl 7-ACA acylase isolated from Bacillus (Brevibacillus) laterosporus. Chu et al. (U.S. Pat. No. 5,766,871) describe a glutaryl 7-ACA acylase isolated from Pseudomonas nitroreducens. Battistel et al., in EP 0525861, describe glutaryl 7-ACA acylases from various Pseudomonas, Bacillus, and Achromobacter species.
A number of enzymes capable of directly catalyzing hydrolysis of cephalosporin C to 7-ACA (Scheme 1, Step C) have been reported. For example, workers at Asahi Chemical (Ichikawa et al., U.S. Pat. No. 4,774,179; Matsuda et al., J. Bact., 1987, 169:5815-5820 and 5821-5826) disclosed strain SE-495 of Pseudomonas diminuta, and strain SE83 of a closely related Pseudomonas species, both of which produce enzymes capable of effecting the direct conversion of cephalosporin C into 7-ACA. Lein, in U.S. Pat. No. 4,981,789 and EP 0283218, reported a cephalosporin C amidase from Arthrobacter viscous. Lein, in EP 0322032, reported a cephalosporin C amidase from Bacillus megaterium, as did Crawford et al., in U.S. Pat. No. 5,104,800 (and divisional U.S. Pat. No. 5,229,247) and EP 0405846. Iwami et al., in U.S. Pat. No. 5,192,678 (and divisional U.S. Pat. No. 5,320,948) and EP 0475652 later disclosed a cephalosporin C acylase from Pseudomonas diminuta N-176 which is capable of carrying out Step C directly, but which is more adept at catalyzing the conversion of glutaryl 7-ACA into 7-ACA. Such enzymes have not yet been shown to be economically viable for production of 7-ACA.
Preparation of recombinant host cells expressing various glutaryl 7ACA amidases has been described by numerous workers. See for example M. Ishiye and M. Niwa, Biochim. Biophys. Acta, 1992, 1132:233-239; Croux et al., EP 0469919; Aramori et al., U.S. Pat. No. 5,310,659; Iwami et al., U.S. Pat. No. 5,192,678; and Honda et al., Biosci. Biotechnol. Biochem., 1997, 61:948-955, all of which are incorporated herein by reference.
In view of the value of 7-ACA as a pharmaceutical intermediate, there exists a need for improved 7-ACA amidases that provide superior results in terms of factors such as enzyme cost, reaction rate and yield, and enzyme stability.